Proteomisk Profilering af makrofager ved 2D elektroforese
Summary
Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.
Abstract
The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.
Introduction
Makrofager er heterogene og plast celler, der er i stand til at erhverve forskellige funktionelle fænotyper. In vivo, disse celler reagerer på en lang række mikro-miljø signalssuch mikrobielle produkter, cytokiner osv. 1. In vitro, pro-inflammatoriske fænotype (M1) af makrofag kan induceres af lipopolysaccharid (LPS) og anti-inflammatoriske fænotype (M2) af visse cytokiner, såsom interleukin-4 (IL-4). Desuden kan makrofager skifte fra et aktiveret M1 M2 fænotype, og omvendt ved specifikke signaler 2.
Afhængigt af fænotype, vil makrofager har forskellige funktioner. M1 makrofager er celler, der producerer proinflammatoriske cytokiner, såsom tumornekrosefaktor α (TNF-α), til at dræbe mikroorganismer eller tumorceller 3. I modsætning hertil M2 makrofager forebygge disse inflammatoriske respons som i sårheling og fibrose ved at producere anti-inflammatory faktorer, såsom TGF-β 3,4.
Humane perifere blod-mononukleære celler fra raske donorer blev isoleret ved Ficoll densitetsgradientcentrifugering som tidligere beskrevet 5 ved anvendelse af en teknik, tilpasset fra Boyum 6. Makrofager i kultur kan differentieres i M1 eller M2 fænotype efter 6 dages primær kultur 7.
Analyse af protein-ekspression eller protein ændringer mellem de to undertyper af makrofager under forskellige kontrollerede stimuli, såsom vært sygdomsfremkaldende virkninger eller mikrobielle toksiner, vil være nyttigt at dechifrere funktionaliteten af pro- og anti-inflammatoriske makrofager.
Proteomics er unikke værktøjer til direkte overvågning af proteiner, der er specifikt op- eller nedreguleret i humane dyrkede makrofager under forskellige stimuli. Fluorescerende farvestoffer har løst nogle af begrænsningerne ved 2D-gelelektroforese, såsom lav følsomhed og billedanalyse 8 < / Sup>. De farvestoffer, der reagerer med cysteinrester har øget detektionsfølsomheden forhold til dem reagere med lysinrester 9. I en tidligere undersøgelse, vi demonstreret nytten af DIGE mætning mærkning til analyse af knappe prøver 10 i forhold til den klassiske sølvfarvet 2D elektroforese 11. Denne teknologi er nyttigt i hurtigt at analysere protein ændringerne mellem de to undertyper af makrofager eller mellem ubehandlede og behandlede makrofager fra samme subtype.
Fordelene ved denne proteomisk teknik har adgang til information af protein størrelse og post-translationelle modifikationer ved at analysere 2D gel 12. Det bør tages i betragtning, at dette ikke er en high throughput teknik, som begrænser antallet af prøver, der kan analyseres. Udviklingen af high throughput assays baseret på massespektrometri som revideret for nylig 13 kan forbedre denne.
_content "> Her præsenterer vi, hvordan du udfører 2D DIGE analyse fra proteinekstraktion af dyrkede makrofager gennem de processer af elektroforese, isoelektrofokusering, og SDS-PAGE samt oplysninger om nytten af tilstrækkelig 2D software.Protocol
Representative Results
Discussion
Den heri beskrevne protokol detaljer en metode til at analysere effekten af forskellige stimuli af de to undertyper af makrofager, M1 (pro-inflammatorisk) og M2 (anti-inflammatorisk). Primære kulturer af M1 og M2 makrofager blev opnået fra differentieringen af monocytter som tidligere publiceret 7..
Fremgangsmåden i 2D DIGE gelelektroforese kræver specialiserede materialer og udstyr, såsom IEF celle til isoelektrofokusering med lav fluorescens plader til SDS-PAGE for at…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by Inserm. Marion Bouvet is a fellow of the French Ministry for Research and Technology. Annie Turkieh is a fellow granted by European Union FP7 HOMAGE (305507).
Materials
| Name | Company | Catalog number |
| RPMI 1640 | Invitrogen | 31870-074 |
| PBX 10 X | Invitrogen | 14200-083 |
| L-glutamine-200mM-100X | Invitrogen | 25030-024 |
| gentamycin 10 mg/mL | Invitrogen | 15710-049 |
| human serum | Invitrogen | 34005100 |
| Ficoll d=1,077 | ATGC | L6115 |
| Leucosep | Dutscher | 16760 |
| 6-wells plate PRIMARIA | Becton Dickinson | 353846 |
| IL-4 | Promocell | B-61410 |
| lipopolysaccharide | Sigma-Aldrich | L-2654 |
| Giemsa | Fluka | 48900 |
| EDTA MM372,2 | Research Organics | 3.00E+01 |
| Filter 0,22µm | Millipore | SCGPTORE |
| 100 mL cylinder | Corning | 430182 |
| TCEP | Interchim | UP242214 |
| Bradford reagent | Bio-Rad | 5000006 |
| Cy3+Cy5-reactive dye | GE Healthcare | 25-8009-83 |
| IPG strip 3-10 24cm | GE Healthcare | 17-6002-44 |
| Protean IEF cell | Bio-Rad | 165-4000 |
| Low-melting agarose | Invitrogen | 15517-014 |
| Ettan-Daltsix system | GE Healthcare | 80-6485-08 |
| Ettan DIGE Imager scanner | GE Healthcare | |
| Progenesis Samespot | Non linear dynamics | |
| 50 ml tubes | any supplier | n/a |
| 15 ml tubes | any supplier | n/a |
| CHAPS | Sigma-Aldrich | C5070 |
| Urée | Merk | 108484-500 |
| Thiourée | Sigma-Aldrich | T7875 |
| DTT | Bio-Rad | 1610611 |
| APS | Sigma-Aldrich | A3678 |
| TEMED | Sigma-Aldrich | T9281 |
| Tris Base | Sigma-Aldrich | T1503 |
| Tris HCl | Sigma-Aldrich | T3253 |
| Pharmalytes 3-10 | GE Healthcare | 17-0456-01 |
| SDS | Sigma-Aldrich | L3773 |
| Bromophenol blue | Sigma-Aldrich | 114391 |
| Glycerol | Sigma-Aldrich | G6279 |
| Acrylamide 40% | Bio-Rad | 161-0148 |
| 2D clean Up | GE Healthcare | 80-6454-51 |
| Glycine | Sigma-Aldrich | G7126 |
| Diméthylformamide | Sigma-Aldrich | 22705-6 |
| electrode wicks | Bio-Rad | 165-4071 |
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